Regulation of phospholipase D activity

ABSTRACT

Novel inhibitors of polyisoprenyl phosphate signaling regulates phopholipase D activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 09/525,157, filed Mar. 14, 2000, now U.S. Pat. No.6,353,026 which in turn claims priority to U.S. Provisional PatentApplication No. 60/125,194, filed Mar. 18, 1999, the contents of whichare incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work leading to this invention was supported in part by NationalInstitutes of Health (NIH) grants GM-38765, DK-50305 and NHLBI-HL-56383.The U.S. Government therefore may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Neutrophil (PMN) activation plays a central role in diverse hostresponses, such as host defense, inflammation and reperfusion injury(Weissmann, G., Smolen, J. E., and Korchak, H. M. (1980) Release ofinflammatory mediators from stimulated neutrophils. N. Engl. J. Med.303, 27-34). In response to inflammatory stimuli, PMN phospholipases areactivated to remodel cell membranes and generate bioactive lipids thatserve as intra- or extracellular mediators in the transduction offunctional responses (Serhan, C. N., Haeggstrom, J. Z., and Leslie, C.C. (1996) Lipid mediator networks in cell signaling: update and impactof cytokines. FASEB J. 10, 1147-1158). Important components ofmicrobicidal and acute inflammatory responses include reactive oxygenspecies and granule enzymes that are targeted to phagocytic vacuoles,but aberrant release of these potentially toxic agents can lead toamplification of inflammation as well as tissue injury and areimplicated in a wide range of diseases (Weiss, S. J. (1989) Tissuedestruction by neutrophils. N. Engl. J. Med. 320, 365-376). To preventan over-exuberant inflammatory response and limit damage to the host,these PMN programs are tightly regulated. The host mediators serving asendogenous anti-inflammatory or protective signals are only recentlybeing appreciated (Serhan, C. N. (1994) Lipoxin biosynthesis and itsimpact in inflammatory and vascular events. Biochim. Biophys. Acta 1212,1-25).

SUMMARY OF THE INVENTION

The present invention pertains to methods for modulating a disease orcondition associated with phospholipase D (PLD) activity. The methodsinclude administration to a subject, an effective anti-PLD amount of alipoxin analog having the formula described infra, such that the PLDinitiated activity is modulated.

The present invention also pertains to methods for treating phosphlipaseD (PLD) activity in a subject. The methods include administration of aneffective anti-PLD amount of a lipoxin analog described infra, such thatPLD initiated activity is treated.

The present invention further pertains to methods for modulating adisease or condition associated with phospholipase D (PLD) initiatedgeneration of superoxide or degranulation activity in a subject. Themethods include, administration of an effective anti-PLD amount of alipoxin analog described infra, such that a disease or conditionassociated with initiated by PLD generation of superoxide ordegranulation activity, is modulated.

The present invention further relates to methods for treatingphospholipase D (PLD) initiated superoxide generation or degranulationactivity in a subject. The methods include administration of aneffective anti-PLD amount of a lipoxin analog described infra, such thatPLD initiated superoxide generation or degranulation activity istreated.

In preferred embodiments, the methods of the invention are performed invitro or in vivo.

In another aspect, the present invention is directed to a packagedpharmaceutical composition for treating the activity or conditionslisted above in a subject. The packaged pharmaceutical compositionincludes a container holding a therapeutically effective amount of atleast one lipoxin compound having one of the formulae described infraand instructions for using the lipoxin compound for treating theactivity or condition in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 shows that LTB₄ rapidly remodels PSDP in human PMN: biosyntheticswitch by an aspirin-triggered LXA₄ analog. Panel A: Scheme foraspirin-triggered 15-epi-LXA₄ biosynthesis and structure of the stableanalog, 15-epi-16-para-fluoro-phenoxy-LXA₄-methyl ester (15-epi-LXa)(left), and hypothetical scheme for PIPP signaling (right). PMN werelabeled with [γ-³²P]-ATP and incubated (12.5×10⁶ ml⁻¹, 37° C.) with LTB₄(●, 100 nM), 15-epi-LXa (♦, 100 nM), vehicle (□, 0.1% ethanol) or15-epi-LXa (100 nM, 5 min) followed by LTB₄ (▴, 100 nM).Non-saponifiable lipids were extracted and separated by TLC, and[³²P]-incorporation was quantitated by phosphoimaging (see Methods).Values are densitometric measurements. Panel B reports a representativetime course (n=5), and Panel C shows the change (mean±S.E.) at 60seconds. *P<0.05 by Student's t-test.

FIG. 2 demonstrates 15-epi-LXA₄ analog inhibits LTB₄-stimulated PLDactivity and superoxide anion generation. Cell lysates (2-5×10⁶ cells,90-130 μg protein) were prepared from the same aliquots of PMN used todetermine PSDP (see FIG. 1 & Methods), warmed to 37° C. and exposed toPC (2 mM in 50 mM Tris-HCl, pH 7.5, plus 30 mM CaCl₂). Reactions wereterminated at 30 sec intervals and choline release was quantitated (26).Values in Panel A are representative (n=5, d=4) of the impact of15-epi-LXa on choline release, and Panel B shows the change at 60seconds (mean±S.E.). Superoxide anion generation by freshly isolatedhuman PMN was determined (10 min, 37° C.) for LTB₄ (100 nM), 15-epi-LXa(100 nM), and increasing concentrations of 15-epi-LXa (1-100 nM, 5 min,37° C.) followed by LTB₄ (100 nM, 10 min, 37° C.). Values reported inPanel C are the mean±S.E. for n=3 separate PMN donors. *P<0.05 byStudent's t-test.

FIG. 3 demonstrates PSDP inhibits phospholipase D. Purified PLD (3 unitsEC 3.1.4.4./125 μl ) was warmed (3 min, 30° C.) and exposed to PSDP(10-1000 nM, 5 min, 30° C.) or vehicle (0.04% ethanol final conc.)followed by PC (0.5-5 mM) in 50 mM Tris-Hcl (pH 7.5) plus 30 mM CaCl₂.Reactions were terminated at 30 sec intervals and choline releasequantitated as in FIG. 2 legend. Values represent the mean for n≧4 forreactions in the absence of PSDP (●, r²=0.963) and the mean for n≧3 withPSDP (

, r²=0.995, 0.971 and 0.953 for 10, 100 and 1000 nM, respectively). CSChem3D Pro software (CambridgeSoft Corp., Cambridge, Mass.) was used tocalculate an energy minimized model of PSDP (inset).

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

It is of wide interest to understand how opposing extracellular signals(positive or negative) are translated into intracellular signalingevents. Receptor-ligand interactions initiate the generation ofbioactive lipids by human neutrophils (PMN) that serve as signals toorchestrate cellular responses important in host defense andinflammation. A novel polyisoprenyl phosphate (PIPP) signaling pathwaywas identified and it was found that one of its components, presqualenediphosphate (PSDP), is a potent negative intracellular signal in PMNthat regulates superoxide anion generation by several stimuli includingphosphatidic acid (Levy et al. (1998) Nature. 389, 985-990). It wasdetermined intracellular PIPP signaling by autacoids with opposingactions on PMN—leukotriene B₄ (LTB₄), a potent chemoattractant, andlipoxin A₄ (LXA₄), a “stop signal” for recruitment. LTB₄ receptoractivation initiated a rapid decrease in PSDP levels concurrent withactivation of PLD and cellular responses. In sharp contrast, activationof the LXA₄ receptor reversed LTB₄-initiated PSDP remodeling leading toan accumulation of PSDP and potent inhibition of both PLD and superoxideanion generation. Thus, an inverse relationship was established for PSDPlevels and PLD activity with two PMN ligands that evoke opposingresponses. In addition, PSDP directly inhibited both isolated humanrecombinant (Ki=6 nM) and plant (Ki=20 nM) PLD. Together, these findingslink PIPP remodeling to intracellular regulation of PMN function andsuggest a role for PIPPs as lipid repressors in signal transduction, anovel mechanism that may also explain aspirin's suppressive actions invivo in cell signaling.

Bioactive lipids are rapidly generated by activation of cell surfacereceptors that carry either specific positive or negative signals tomodulate cellular responses. This is exemplified by the relatedeicosanoids, leukotriene B₄ (LTB₄), a potent chemoattractant (Borgeat,P., and Naccache, P. H. (1990) Biosynthesis and biological activity ofleukotriene B₄ . Clin. Biochem. 23, 459-468), and lipoxin A₄ (LXA₄), anendogenous “stop signal” for PMN recruitment (Serhan, C. N. (1994)Lipoxin biosynthesis and its impact in inflammatory and vascular events.Biochim. Biophys. Acta 1212, 1-25). LTB₄ and LXA₄ interact with highlyspecific and distinct G protein-coupled membrane receptors (Yokomizo,T., Izumi, T., Chang, K., Takuwa, T., and Shimizu, T. (1997) AG-protein-coupled receptor for leukotriene B₄ that mediates chemotaxis.Nature 387, 620-624; Fiore, S., Romano, M., Reardon, E. M., and Serhan,C. N. (1993) Induction of functional lipoxin A₄ receptors in HL-60cells. Blood 81, 3395-3403). They each evoke opposing PMN responses,including LXA₄ inhibition of LTB₄-initiated chemotaxis, adhesion andtransmigration (Serhan, C. N. (1994) Lipoxin biosynthesis and its impactin inflammatory and vascular events. Biochim. Biophys. Acta 1212, 1-25).

Abbreviations used throughout this application include: COX,cyclooxygenase; 15-epi-LX, 15-epimer lipoxin; 15-epi-LXa,15-epi-16-para-fluoro-phenoxy LXA₄-methyl ester; FDP, farnesyldiphosphate; GST, glutathione-S-transferase; LTB₄, leukotriene B₄; LO,lipoxygenase; PA, phosphatidic acid; PC, phosphatidychloine; cPLD,cabbage phospholipase D; PIPP, polyisoprenyl phosphate; PMN,polymorphonuclear leukocytes; PSDP, presqualene diphosphate; PSMP,presqualene monophosphate; Sf9, Spodoptera frugiperda; TLC, thin-layerchromatography.

Aspirin is known to affect biosynthesis of lipid mediators and is widelyused clinically for its anti-inflammatory properties. Mechanismsresponsible for aspirin's anti-inflammatory actions remain ofconsiderable interest. In particular, new “super-aspirins” are soughtthat spare the gastrointestinal tract and do not possess the deleteriousside effects of steroids (Isakson, P., Seibert, K., Masferrer, J.,Salvemini, D., Lee, L., and Needleman, P. (1995) Discovery of a betteraspirin. Advances in Prostaglandin, Thromboxane & Leukotriene Research23, 49-54). In one aspect it has been that, in addition to inhibitingprostanoid formation, aspirin triggers the endogenous generation ofnovel carbon 15 epimers of LX by transcellular routes (see FIG. 1A)during inflammation in vivo (e.g., between tissue resident cells andinfiltrating leukocytes) (Chiang, N., Takano, T., Clish, C. B., Petasis,N. A., Tai, H.-H., and Serhan, C. N. (1998) Aspirin-triggered15-epi-lipoxin A₄ (ATL) generation by human leukocytes and murineperitonitis exudates: development of a specific 15-epi-LXA₄ ELISA. J.Pharmacol Exper. Ther. 287, 779-790). These aspirin-triggered lipoxins(15-epi-LX) are even more potent than the native LX as inhibitors of PMNresponses, in part because they are active longer (Serhan, C. N.,Maddox, J. F., Petasis, N. A., Akritopoulou-Zanze, I., Papayianni, A.,Brady, H. R., Colgan, S. P., and Madara, J. L. (1995) Design of lipoxinA₄ stable analogs that block transmigration and adhesion of humanneutrophils. Biochemistry 34, 14609-14615). PMN inhibition by LX and15-epi-LX is evoked by specific receptor-activation of “inhibitory”signals and not via direct receptor level antagonism at LTB₄ receptors(Takano, T., Fiore, S., Maddox, J. F., Brady, H. R., Petasis, N. A., andSerhan, C. N. (1997) Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA₄Stable analogues are potent inhibitors of acute inflammation: Evidencefor anti-inflammatory receptors. J. Exp. Med. 185, 1693-1704). Moreover,interest in the regulation of the LTB₄ receptor is heightened by therecent finding that LTB₄ receptors also serve as novel HIV-1 coreceptors(Owman, C., Garzino-Demo, A., Cocchi, F., Popovic, M., Sabirsh, A., andGallo, R. (1998) The leukotriene B₄ receptor functions as a novel typeof coreceptor mediating entry of primary HIV-1 isolates intoCD4-positive cells. Proc. Natl. Acad. Sci. 95, 9530-9534).

Despite ˜100 years of use, complete knowledge of aspirin's therapeuticimpact is still evolving with many newly discovered clinical utilities(Marcus, A. J. (1995) Aspirin as prophylaxis against colorectal cancer.N. Engl. J. Med. 333, 656-658). Regular ingestion of aspirin decreasesthe incidence of myocardial infarction, colorectal carcinoma andAlzheimer's disease (Vainio, H., and Morgan, G. (1997) Aspirin for thesecond hundred years: new uses for an old drug. Pharmacol Toxicol 81,151-152), but side effects from aspirin, such as gastrointestinalulceration, can limit its use. The recent discovery of a second isoformof cyclooxygenase (COX) that is induced during inflammation has led to asearch for “super-aspirins” that can selectively inhibit COX-2 withoutdisrupting the protective constitutive functions of COX-1 (Isakson, P.,Seibert, K., Masferrer, J., Salvemini, D., Lee, L., and Needleman, P.(1995) Discovery of a better aspirin. Advances in Prostaglandin,Thromboxane & Leukotriene Research 23, 49-54; Herschman, H. R. (1998)Recent progress in the cellular and molecular biology of prostaglandinsynthesis. Trends in Cardiovasc. Med. 8, 145-150). Of particularinterest in this regard, 15-epi-LX, which inhibit PMN migration, areendogenous products of aspirin's acetylating ability that may underlysome of the salutary benefits of aspirin. Both LX and 15-epi-LX stableanalogs were prepared, which like 15-epi-LXA₄, act via the LXA₄ receptor(Serhan, C. N., Maddox, J. F., Petasis, N. A., Akritopoulou-Zanze, I.,Papayianni, A., Brady, H. R., Colgan, S. P., and Madara, J. L. (1995)Design of lipoxin A₄ stable analogs that block transmigration andadhesion of human neutrophils. Biochemistry 34, 14609-14615; Takano, T.,Fiore, S., Maddox, J. F., Brady, H. R., Petasis, N. A., and Serhan, C.N. (1997) Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA₄ Stableanalogues are potent inhibitors of acute inflammation: Evidence foranti-inflammatory receptors. J. Exp. Med. 185, 1693-1704). Suitablemethods of preparation of lipoxin compounds can also be found, forexample, in U.S. Pat. Nos. 5,411,951, 5,648,512, 5,650,435 and5,750,354, incorporated herein by reference. For example,15-epi-16-para-fluoro-phenoxy-lipoxin A₄-methyl ester (15-epi-LXa) is asynthetic analog of 15-epi-LXA₄ (FIG. 1A, bottom left) that not onlyresists rapid inactivation but acts topically to inhibit PMNinfiltration and vascular permeability in mouse ear skin inflammation(Takano, T., Clish, C. B., Gronert, K., Petasis, N., and Serhan, C. N.(1998) Neutrophil-mediated changes in vascular permeability areinhibited by topical application of aspirin-triggered 15-epi-lipoxin A₄and novel lipoxin B₄ stable analogues. J. Clin. Invest. 101, 819-826).

Elucidation of signaling pathway(s) responsible for receptor-operatedblockage of PMN responses is of interest. Signaling via phospholipase D(PLD) plays a pivotal role in mounting cellular responses. Withinseconds of exposure to ligands, PLD hydrolyzes membranephosphatidylcholine (PC) to generate phosphatidic acid (PA)(Billah, M.M., Eckel, S., Mullmann, T. J., Egan, R. W., and Siegel, M. I. (1989)Phosphatidylcholine hydrolysis by phospholipase D determinesphosphatidate and diglyceride levels in chemotactic peptide-stimulatedhuman neutrophils. Involvement of phsophatidate phosphohydrolase insignal transduction. J. Biol. Chem. 264, 17069-17077). Formation of PAtemporally antecedes functional responses, including vesicle secretionand assembly of the NADPH oxidase (Wakelam, M. J. O., Martin, A.,Hodgkin, M. N., Brown, F., Pettit, T. R., Cross, M. J., De Takats, P.G., and Reynolds, J. L. (1997) Role and regulation of phospholipase Dactivity in normal and cancer cells. Advances in Enzyme Regulation 37,29-34; Olson, S. C., and Lambeth, J. D. (1996) Biochemistry and cellbiology of phospholipase D in human neutrophils. Chem. Phys. Lipids 80,3-19). Several isozymes of PLD1 and PLD2 were cloned and characterized(Steed, P. M., Clark, K. L., Boyar, W. C., and Lasala, D. J. (1998)Characterization of human PLD2 and the analysis of PLD isoform splicevariants. FASEB J. 12, 1309-1317), with PLD1b identified as a prominentisoform in human granulocytes (Martin, A., Saqib, K. M., Hodgkin, M. N.,Brown, F. D., Pettit, T. R., Armstrong, S., and Wakelam, M. J. O. (1997)Role and regulation of phospholipase D signalling. Biochem. Soc. Trans.25, 1157-1160). The complete DNA and amino acid sequences for human PLDis disclosed in Hammond et al. (1995) J. Biol. Chem. 270: 29640-29643,and Hammond et al. (1997) J. Biol. Chem. 272: 3860-3868, the entirecontents of which are incorporated herein by reference, and can also befound at GenBank Accession Nos. NM 002662 and U38545.

Recently, a novel polyisoprenyl phosphate (PIPP) signaling pathway wasidentified (FIG. 1A) and found that, in PMN, presqualene diphosphate(PSDP) carries biological activity and serves as a negativeintracellular signal that prevents superoxide anion generation byseveral stimuli including PA (Levy, B. D., Petasis, N. A., and Serhan,C. N. (1997) Polyisoprenyl phosphates in intracellular signalling.Nature 389, 985-989). Because PLD activation is linked to superoxideanion generation (Agwu, D. E., McPhail, L. C., Sozzani, S., Bass, D. A.,and McCall, C. E. (1991) Phosphatidic acid as a second messenger inhuman polymorphonuclear leukocytes. Effects on activation of NADPHoxidase. J. Clin. Invest. 88, 531-539), it was determined that PIPPsignaling also modulates phospholipase activity critical to globalcellular activation. It was found that (i) that LTB₄ receptor activationrapidly degrades PSDP, a key component of PIPP signaling, that isreversed by a LXA₄ receptor agonist, (ii) that an aspirin-triggered15-epi-LXA₄ stable analog potently inhibits LTB₄-initiated PLDactivation and superoxide anion generation, and (iii) that PSDP directlyinhibits both human recombinant and plant PLD. These findings provideevidence for receptor-initiated PIPP remodeling as a regulatorysignaling pathway.

The present invention pertains to methods for modulating a disease orcondition associated with phospholipase D (PLD) activity. The methodsinclude administration to a subject, an effective anti-PLD amount of alipoxin analog having the formula described infra, such that the PLDinitiated activity is modulated.

The present invention also pertains to methods for treating phosphlipaseD (PLD) activity in a subject. The methods include administration of aneffective anti-PLD amount of a lipoxin analog described infra, such thatPLD initiated activity is treated.

The present invention further pertains to methods for modulating adisease or condition associated with phospholipase D (PLD) initiatedgeneration of superoxide or degranulation activity in a subject. Themethods include, administration of an effective anti-PLD amount of alipoxin analog described infra, such that a disease or conditionassociated with initiated by PLD generation of superoxide ordegranulation activity, is modulated.

The present invention further relates to methods for treatingphospholipase D (PLD) initiated superoxide generation or degranulationactivity in a subject. The methods include administration of aneffective anti-PLD amount of a lipoxin analog described infra, such thatPLD initiated superoxide generation or degranulation activity istreated.

In preferred embodiments, the methods of the invention are performed invitro or in vivo.

In another aspect, the present invention is directed to a packagedpharmaceutical composition for treating the activity or conditionslisted above in a subject. The packaged pharmaceutical compositionincludes a container holding a therapeutically effective amount of atleast one lipoxin compound having one of the formulae described infraand instructions for using the lipoxin compound for treating theactivity or condition in the subject.

In one embodiment, compounds useful in the invention have the formula(I)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein Q₃ and Q₄ are each independently O, S or NH;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a =0 to 3,        b=0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;    -   wherein T is O or S, and pharmaceutically acceptable salts        thereof.

In another embodiment, compounds useful in the invention have theformula (II)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR₁, wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3,        b=0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;    -   wherein T is O or S, and pharmaceutically acceptable salts        thereof.

The invention is also directed to useful lipoxin compounds having theformula (III)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;    -   wherein T is O or S, and pharmaceutically acceptable salts        thereof.

The invention is further directed to useful lipoxin compounds having theformula (IV)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;    -   wherein T is O or S, and pharmaceutically acceptable salts        thereof.

The invention is further directed to useful lipoxin compounds having theformula (V)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO_(2,) —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched; and    -   pharmaceutically acceptable salts thereof.

In preferred embodiments, X is OR₁ wherein R₁ is a hydrogen atom, analkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptablesalt, Q₁ is C═O, R₂ and R₃, if present, are hydrogen atoms, R₄ is ahydrogen atom or methyl, Q₃ and Q₄, if present, are both O, R₆, ifpresent, is a hydrogen atom, Y₁, if present, is OH, T is O and R₅ is asubstituted phenyl, e.g.,

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl. In certain embodiments for R₅,        para-fluorophenyl and/or unsubstituted phenyl are excluded,        e.g., 15-epi-16-(para-fluoro)-phenoxy-LXA₄,        16-(para-fluoro)-phenoxy-LXA₄, 15-epi-16-phenoxy-LXA₄ or        16-phenoxy-LXA₄. The compounds encompassed by U.S. Pat. No.        5,441,951 are excluded from certain aspects of the present        invention.

In still another aspect, the present invention is directed topharmaceutical compositions including compounds having theabove-described formulae and a pharmaceutically acceptable carrier. Inone embodiment, a preferred compound is

In a preferred embodiment, the pharmaceutical carrier is not a ketone,e.g., acetone.

In preferred embodiments, Y₁ is a hydroxyl and the carbon bearing thehydroxyl can have an R or S configuration. In most preferredembodiments, the chiral carbon bearing the hydroxyl group, e.g., Y₁ isdesignated as a 15-epi-lipoxin as is known in the art.

In certain embodiments the chirality of the carbons bearing the R₂, R₃,Q₃ and Q₄ groups can each independently be either R or S. In preferredembodiments, Q₃ and Q₄ have the chiralities shown in structures II, III,IV or V.

In preferred embodiments, R₄ is a hydrogen. In other preferredembodiments, R₆ is a hydrogen.

Additionally, R₅ can be a substituted or unsubstituted, branched orunbranched alkyl group having between 1 and about 6 carbon atoms,preferably between 1 and 4 carbon atoms, most preferably between 1 and3, and preferably one or two carbon atoms. The carbon atoms can havesubstituents which include halogen atoms, hydroxyl groups, or ethergroups.

The compounds useful in the present invention can be prepared by thefollowing synthetic scheme:

-   -   wherein X, Q₁, Q₃, Q₄, R₂, R₃, R₄, R₅, R₆, Y₁ and T are as        defined above. Suitable methods known in the art to can be used        to produce each fragment. For example, the acetylenic fragment        can be prepared by the methods discussed in Nicolaou, K. C. et        al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100; Nicolaou, K. C.        et al. (1989) J. Org. Chem. 54:5527; Webber, S. E. et al. (1988)        Adv. Exp. Med. Biol. 229:61; and U.S. Pat. No. 5,441,951. The        second fragment can be prepared by the methods of Raduchel, B.        and Vorbruggen, H. (1985) Adv. Prostaglandin Thromboxane        Leukotriene Res. 14:263.

A “lipoxin analog” shall mean a compound which has an “active region”that functions like the active region of a “natural lipoxin”, but whichhas a “metabolic transformation region” that differs from naturallipoxin. Lipoxin analogs include compounds which are structurallysimilar to a natural lipoxin, compounds which share the same receptorrecognition site, compounds which share the same or similar lipoxinmetabolic transformation region as lipoxin, and compounds which areart-recognized as being analogs of lipoxin. Lipoxin analogs includelipoxin analog metabolites. The compounds disclosed herein may containone or more centers of asymmetry. Where asymmetric carbon atoms arepresent, more than one stereoisomer is possible, and all possibleisomeric forms are intended to be included within the structuralrepresentations shown. Optically active (R) and (S) isomers may beresolved using conventional techniques known to the ordinarily skilledartisan. The present invention is intended to include the possiblediastereiomers as well as the racemic and optically resolved isomers.

The terms “corresponding lipoxin” and “natural lipoxin” refer to anaturally-occurring lipoxin or lipoxin metabolite. Where an analog hasactivity for a lipoxin-specific receptor, the corresponding or naturallipoxin is the normal ligand for that receptor. For example, where ananalog is a LXA₄ specific receptor on differentiated HL-60 cells, thecorresponding lipoxin is LXA₄. Where an analog has activity as anantagonist to another compound (such as a leukotriene), which isantagonized by a naturally-occurring lipoxin, that natural lipoxin isthe corresponding lipoxin.

“Active region” shall mean the region of a natural lipoxin or lipoxinanalog, which is associated with in vivo cellular interactions. Theactive region may bind the “recognition site” of a cellular lipoxinreceptor or a macromolecule or complex of macromolecules, including anenzyme and its cofactor. Preferred lipoxin A₄ analogs have an activeregion comprising C₅-C₁₅ of natural lipoxin A₄. Preferred lipoxin B₄analogs have an active region comprising C₅-C₁₄ of natural lipoxin B4.

The term “recognition site” or receptor is art-recognized and isintended to refer generally to a functional macromolecule or complex ofmacromolecules with which certain groups of cellular messengers, such ashormones, leukotrienes, and lipoxins, must first interact before thebiochemical and physiological responses to those messengers areinitiated. As used in this application, a receptor may be isolated, onan intact or permeabilized cell, or in tissue, including an organ. Areceptor may be from or in a living subject, or it may be cloned. Areceptor may normally exist or it may be induced by a disease state, byan injury, or by artificial means. A compound of this invention may bindreversibly, irreversibly, competitively, noncompetitively, oruncompetitively with respect to the natural substrate of a recognitionsite.

The term “metabolic transformation region” is intended to refergenerally to that portion of a lipoxin, a lipoxin metabolite, or lipoxinanalog including a lipoxin analog metabolite, upon which an enzyme or anenzyme and its cofactor attempts to perform one or more metabolictransformations which that enzyme or enzyme and cofactor normallytransform on lipoxins. The metabolic transformation region may or maynot be susceptible to the transformation. A nonlimiting example of ametabolic transformation region of a lipoxin is a portion of LXA₄ thatincludes the C-13,14 double bond or the C-15 hydroxyl group, or both.

The term “detectable label molecule” is meant to include fluorescent,phosphorescent, and radiolabeled molecules used to trace, track, oridentify the compound or receptor recognition site to which thedetectable label molecule is bound. The label molecule may be detectedby any of the several methods known in the art.

The term “labeled lipoxin analog” is further understood to encompasscompounds which are labeled with radioactive isotopes, such as but notlimited to tritium (³H), deuterium (²H), carbon (¹⁴C), or otherwiselabeled (e.g. fluorescently). The compounds of this invention may belabeled or derivatized, for example, for kinetic binding experiments,for further elucidating metabolic pathways and enzymatic mechanisms, orfor characterization by methods known in the art of analyticalchemistry.

The term “inhibits metabolism” means the blocking or reduction ofactivity of an enzyme which metabolizes a native lipoxin. The blockageor reduction may occur by covalent bonding, by irreversible binding, byreversible binding which has a practical effect of irreversible binding,or by any other means which prevents the enzyme from operating in itsusual manner on another lipoxin analog, including a lipoxin analogmetabolite, a lipoxin, or a lipoxin metabolite.

The term “resists metabolism” is meant to include failing to undergo oneor more of the metabolic degradative transformations by at least one ofthe enzymes which metabolize lipoxins. Two nonlimiting examples of LXA₄analog that resists metabolism are 1) a structure which can not beoxidized to the 15-oxo form, and 2) a structure which may be oxidized tothe 15-oxo form, but is not susceptible to enzymatic reduction to the13,14-dihydro form.

The term “more slowly undergoes metabolism” means having slower reactionkinetics, or requiring more time for the completion of the series ofmetabolic transformations by one or more of the enzymes which metabolizelipoxin. A nonlimiting example of a LXA₄ analog which more slowlyundergoes metabolism is a structure which has a higher transition stateenergy for C-15 dehydrogenation than does LXA₄ because the analog issterically hindered at the C-16.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “halogen” is meant to include fluorine, chlorine, bromine andiodine, or fluoro, chloro, bromo, and iodo. In certain aspects, thecompounds of the invention do not include halogenated compounds, e.g.,fluorinated compounds.

The term “subject” is intended to include living organisms susceptibleto conditions or diseases caused or contributed to by inflammation,inflammatory responses, vasoconstriction, and myeloid suppression.Examples of subjects include humans, dogs, cats, cows, goats, and mice.The term subject is further intended to include transgenic species.

When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

In certain embodiment, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts” in theseinstances refers to the relatively non-toxic, inorganic and organic baseaddition salts of compounds of the present invention. These salts canlikewise be prepared in situ during the final isolation and purificationof the compounds, or by separately reacting the purified compound in itsfree acid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters. In a preferred embodiment, the ester is not amethyl ester (See, for example, Berge et al., supra.).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred per cent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous injection administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 0.1 to about 40 mg per kg per day. For example,between about 0.01 microgram and 20 micrograms, between about 20micrograms and 100 micrograms and between about 10 micrograms and 200micrograms of the compounds of the invention are administered per 20grams of subject weight.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

Methods

Materials.

15-epi-LXa, PSDP and PSMP were prepared by total organic synthesis andcharacterized by their physical chemical and biological properties(Takano, T., Clish, C. B., Gronert, K., Petasis, N., and Serhan, C. N.(1998) Neutrophil-mediated changes in vascular permeability areinhibited by topical application of aspirin-triggered 15-epi-lipoxin A₄and novel lipoxin B₄ stable analogues. J. Clin. Invest. 101, 819-826;Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997) Polyisoprenylphosphates in intracellular signaling. Nature 389, 985-989). LTB₄ waspurchased from Cayman Chemical (Ann Arbor, Mich.), cabbage PLD (cPLD),FDP, squalene, lysis buffer reagents and cytochrome c were from SigmaChemical Co. (St. Louis, Mo.), and PC and PA were from Avanti PolarLipids (Alabaster, Ala.). The integrity and concentration of eachbioactive lipid was assessed just prior to each series of experiments byUV analysis (eicosanoids and analogs) and phosphorus determinations(polyisoprenyl phosphates) (Takano, T., Clish, C. B., Gronert, K.,Petasis, N., and Serhan, C. N. (1998) Neutrophil-mediated changes invascular permeability are inhibited by topical application ofaspirin-triggered 15-epi-lipoxin A₄ and novel lipoxin B₄ stableanalogues. J. Clin. Invest. 101, 819-826; Levy, B. D., Petasis, N. A.,and Serhan, C. N. (1997) Polyisoprenyl phosphates in intracellularsignaling. Nature 389, 985-989).

Human PMN

Peripheral venous blood (˜180 ml) was obtained by venipuncture fromhealthy volunteers who denied taking any medication for at least twoweeks and had given written informed consent to a protocol approved byBrigham and Women's Hospital's Human Research Committee. PMN wereisolated from whole blood and steady state labeled with [γ-³²P]ATP (40μCiml⁻¹, 90 min, 37° C.) as in (Levy, B. D., Petasis, N. A., and Serhan,C. N. (1997) Polyisoprenyl phosphates in intracellular signalling.Nature 389, 985-989). Labeled PMN were resuspended (20×10⁶ ml⁻¹ PBS with1 mM CaCl₂, pH 7.40) and exposed to LTB₄ (100 nM), 15-epi-LXa (100 nM)or vehicle (0.1% EtOH) for 0 to 300 seconds (37° C.). From eachincubation, aliquots were removed at indicated intervals to determinethe radiolabeling of nonsaponifiable lipids (10-12×10⁶ PMN) and PLDactivity (1″1.25×10⁶ PMN). Materials present in each incubation weresaponified, extracted and separated by TLC with phosphoimaging (model425E and integration software; Molecular Dynamics), which was used forPSDP mass determination as in ref 22.

Preparation of Recombinant Human PLD1b.

Spodoptera frugiperda (Sf9) cells were cultured in suspension at 2×10⁵to 2×10⁶ cells/ml TC100 medium supplemented with 10% fetal calf serum(Gibco). A cDNA encoding human PLD1b (cloned from placental tissue) wasinserted into the transfer vector pACGHLT (Pharmingen) downstream of,and in frame with, vector sequences encoding glutathione-S-transferase(GST), hexahistidine, a protein kinase A phosphorylation site and athrombin cleavage site. The GST-hPLD1b construct was cotransfected intoSf9 cells with linearized, polyhedrin-minus (PH-), AcMNPV DNA,Bac-N-Blue, according to the supplier's instructions (Invitrogen).Homologous recombination between linearized virus and the transfervector restored the function of essential viral gene ORF1629 to yieldinfectious, recombinant virus. After two rounds of plaque-purification,recombinant virus was amplified by large scale infections of Sf9 cellsuntil a titer of 8×10⁷ pfu/ml was obtained. To generate GST-hPLD1b, 500ml of Sf9 cells at 2×10⁶ cells/ml were infected with virus at amultiplicity of infection of 10:1. Cells were harvested 72 hourspost-infection, lysed and the expressed GST-hPLD1b purified onglutathione agarose beads, according to supplier's instructions(Pharmingen). The purified recombinant protein was identified byimmunoreactivity with goat anti-GST pAb (Amersham Pharmacia Biotech) andrabbit pAb raised against the PLD consensus peptide sequence GSANIN(gift of P. Parker, ICRF, London, UK), and by activity in an in vitroPLD assay (24).

PLD Activity and Superoxide Anion Generation.

Lysates were generated from cells at rest or after exposure to agonistusing a lysis buffer comprised of 0.1 M Hepes (pH 7.4), 0.7 mM sodiumorthovanadate, 10 μM p-nitrophenylphosphate, 10 mM EGTA, 5.5% tritonX-100, 0.5 M β-glycerophosphate, 10 mM phenylmethylsulfonylfluoride, 0.1mM ammonium molybdate, 12 mM DFP, 5 μgml⁻¹ leupeptin, 2 μgml⁻¹ aprotininand 7 μgml⁻¹ pepstatin A (as in ref 25) and utilized for bioassay.

PMN lysates (90-130 μg protein), purified phospholipase D (3-30 units)(EC 3.1.4.4., Sigma Chemical Co.) or recombinant hPLD1b were warmed (37°C. for mammalian enzyme and 30° C. for cabbage, 3 min) and exposed toPSDP, PSMP or FDP (10-1000 μM, 5 min, 37° C. or 30° C.) followed by PC(0.5 to 5 mM) in Tris-HCl (50 mM, pH 7.5) with CaCl₂ (30 mM). Reactionswere terminated at 30 second intervals (0-90 seconds) with Tris-HCl (1M) plus EDTA (50 mM). Choline release was quantitated as in ref 26.

Freshly isolated human PMN (1-3×10⁶ PMN/ml HBSS+1.6 mM CaCl₂) wereincubated (5 min, 37° C.) in the presence of 15-epi-LXa (1-100 nM) orvehicle (0.1% ethanol), and then exposed (10 min) to LTB₄ (100 nM) inthe presence of cytochrome c (7 mg/ml). Superoxide anion generation wasdetermined as in ref 22.

Statistical Analysis.

Results are expressed as the mean±S.E. and statistical significance wasevaluated using the Student's test.

Results

Leukotriene B₄ Stimulates Rapid Remodeling of PIPP: Degradation of PSDP.

Leukotriene B₄ interacts with its receptor to rapidly activatephospholipases and signal cellular responses (Yokomizo, T., Izumi, T.,Chang, K., Takuwa, T., and Shimizu, T. (1997) A G-protein-coupledreceptor for leukotriene B₄ that mediates chemotaxis. Nature 387,620-624). To determine if LTB₄ receptor activation lead to remodeling ofPIPP and specifically PSDP, cellular phosphate pools were steady statelabeled with [γ-³²P]-ATP (see Methods) and exposed to either LTB₄ (100nM) or vehicle (0.1% ethanol) alone. Aliquots were removed at timedintervals from 0 to 300 sec (37° C.) and non-saponifiable phosphorylatedlipids were isolated and quantitated by phosphoimager for [³²P]incorporation. PSDP levels in unstimulated PMN are ˜1.7 nmoles/10⁷ PMN(˜50 nM) (Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997)Polyisoprenyl phosphates in intracellular signalling. Nature 389,985-989). PSDP and presqualene monophosphate (PSMP), but not farnesyldiphosphate (FDP), incorporated [³²P] from ATP, consistent with recentevidence (Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997)Polyisoprenyl phosphates in intracellular signalling. Nature 389,985-989). LTB₄ initiated a rapid (evident within 30 sec) (FIG. 1B) andstatistically significant decrease in [³²P]-PSDP (28%) within 60 sec(FIG. 1C). Within the ensuing 270 sec, [³²P]-PSDP levels returned tobaseline amounts (t=0). Changes in [³²P]-PSDP after LTB₄ receptoractivation reflected changes in PSDP mass. These results confirmed thatPSDP was present in PMN (Levy, B. D., Petasis, N. A., and Serhan, C. N.(1997) Polyisoprenyl phosphates in intracellular signalling. Nature 389,985-989) and indicated that LTB₄ initiated a marked decrement in PSDP(FIG. 1) with a time course of PIPP remodeling concurrent with LTB₄kinetics of cellular activation (Borgeat, P., and Naccache, P. H. (1990)Biosynthesis and biological activity of leukotriene B₄ . Clin. Biochem.23, 459-468; Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997)Polyisoprenyl phosphates in intracellular signalling. Nature 389,985-989).

15-epimer LX Analog Switches the LTB₄ Program to Enhance PSDP.

Both LXA₄ and some 15-epi-LXA₄ stable analogs act at the LXA₄ receptoron PMN, inhibiting infiltration in vivo. To determine if LX and15-epi-LX mediate inhibitory actions via PIPP signaling, the impact of a15-epi-LXA₄ analog (15-epi-LXa) (100 nM, 5 min, 37° C.) on LTB₄ (100nM)-stimulated changes in PSDP was examined using [³²P] labeling of PMNlipids (vide supra, in parallel incubations). Alone, 15-epi-LXa did notaffect the rate of PIPP remodeling (FIG. 1B). Of interest, exposure toLTB₄ in the presence of equimolar 15-epi-LXa not only prevented the LTB₄initiated decrease in PSDP, but additionally stimulated a significantincrease (˜72%) in [³²P]-PSDP at 60 sec (FIG. 1C). PSDP levels continuedto rise for at least 300 sec after exposure to LTB₄ (FIG. 1B). NativeLXA₄ and its related LXA₄ receptor agonist, 16-phenoxy-LXA₄-methylester, gave qualitatively similar responses as 15-epi-LXa with a rankorder of potency of 15-epi-LXa>16-phenoxy-LXA₄>LXA₄ with 15-epi-LXa 1-2orders of magnitude more potent. These results indicate that 15-epi-LXa,which inhibits LTB₄ responses in vivo (Takano, T., Fiore, S., Maddox, J.F., Brady, H. R., Petasis, N. A., and Serhan, C. N. (1997)Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA₄ Stable analogues arepotent inhibitors of acute inflammation: Evidence for anti-inflammatoryreceptors. J. Exp. Med. 185, 1693-1704), dramatically switchesLTB₄-initiated PIPP signaling. Moreover, increases in PSDP levels evokedby coactivation of the LXA₄ and LTB₄ receptors indicate that the timecourse of PSDP accumulation correlated with regulation of LTB₄'s actionsby LX and 15-epi-LXa (vide infra).

15-epi-LXa Inhibits LTB₄-stimulated PLD Activity and O₂ Generation.

LTB₄-stimulated PLD activity is associated with morphologic change,degranulation and O₂ ⁻ production in PMN (Olson, S. C., and Lambeth, J.D. (1996) Biochemistry and cell biology of phospholipase D in humanneutrophils. Chem. Phys. Lipids 80, 3-19; Zhou, H.-L., Chabot-Fletcher,M., Foley, J. J., Sarau, H. M., Tzimas, M. N., Winkler, J. D., andTorphy, T. J. (1993) Association between leukotriene B₄-inducedphospholipase D activation and degranulation of human neutrophils.Biochem. Pharmacol. 46, 139-148). To determine whether LT andLX-mediated remodeling of PIPP correlates with specific cell signalingevents, PLD activity was monitored in cell lysates from the sameincubations used in FIG. 1. LTB₄ gave increases in PLD activity thatwere maximal by 60 sec (FIG. 2A). These values for LTB₄ and PLD areconsistent with those of earlier reports (Gomez-Cambronero, J. (1995)Immunoprecipitation of a phospholipase D activity withantiphosphotyrosine antibodies. J. Interferon Cytokine Res. 15, 877-885;Zhou, H.-L., Chabot-Fletcher, M., Foley, J. J., Sarau, H. M., Tzimas, M.N., Winkler, J. D., and Torphy, T. J. (1993) Association betweenleukotriene B₄-induced phospholipase D activation and degranulation ofhuman neutrophils. Biochem. Pharmacol. 46, 139-148). In the presence of15-epi-LXa, LTB₄-stimulated PLD activity was inhibited (˜81%) at 60 sec(FIGS. 2A&B). 15-epi-LXa also potently inhibited LTB₄-stimulated O₂ ⁻generation (FIG. 2C). Together, these findings indicate thatligand-receptor interaction that signals opposing cellular responsesgave an inverse relationship between [³²P]-PSDP levels and PLD activity,demonstrating that PSDP could regulate PLD.

Direct Inhibition of both Plant and Mammalian PLD.

To determine whether polyisoprenyl phosphates act directly on PLD, PSDPand closely related lipids were incubated with purified plant enzyme (EC3.1.4.4; Vm=0.29 mmoles/sec, Km=1.4 mM). As seen in FIG. 3, PSDPinhibited cPLD in a concentration-dependent fashion (10 to 1000 nM) witha Ki of 20 nM ((PSDP) =10 nM). Lineweaver-burk analyses (FIG. 3) wereconsistent with a competitive inhibition model. Closely related lipids,such as PSMP (minus only one phosphate), showed a greater than 100-foldloss in inhibitory potency compared to PSDP (Table I). Comparableinhibition was not evident with other polyisoprenoids (i.e., FDP andsqualene) or a PLD product (PA). It was determined whether PSDP couldalso inhibit mammalian PLD by determining recombinant human PLD1bkinetics in vitro with PSDP. The recombinant enzyme (Vm=0.36 nmoles/sec,Km=13.8 mM) was also dramatically inhibited by PSDP with a Ki of 6 nM(Table I).

TABLE 1 PSDP selectively inhibits phospholipase D: structure activityrelationship with related endogenous lipids^(a) Lipid Enzyme K_(m) (mM)V_(m) cPLD 1.4 0.29 rhPLD1b 13.8  0.36 K_(m app) (mM) V_(m app) K_(i)(nM) Presqualene diphosphate cPLD 2.1 0.25 20  rhPLD1b 3.1 0.03 6Presqualene cPLD 3.1 0.36 3210   monophosphate Squalene cPLD 4.0 0.46 0Farnesyl diphosphate cPLD 0.9 0.25 0 Phosphatidic acid cPLD 3.6 0.43 0^(a)Enzyme kinetics for PLD were determined in the presence of PSDP orrelated lipids. Purified or isolated PLD (3u cPLD or 0.3urhPLD1b/reaction) activity in the presence of the test compounds wasdetermined as described in FIG. 3 legend (n ≧ 3). K, was calculatedusing the formula: Slope = K_(m)/V_(m) (1 + [I]/K_(i)). Absence ofinhibition is reported as K, =0.

Because PLD activation occurs in vivo in the presence of many cofactorswhich modulate its activity, it was also determined the impact of PSDPon PLD activity in PMN lysates. Sixty seconds following LTB₄, PSDPlevels decreased (28%, FIG. 1) and PLD activity was maximal (FIG. 2).Addition of PSDP (100 nM) to PMN lysates at this time (60 sec, LTB₄ 100nM) gave 89.5+/−9.7% inhibition of PLD activity. Collectively, theseresults indicated that PSDP was a potent inhibitor of both plant andmammalian PLD's and establish a critical role for both the terminalphosphate and the isoprenoid chain length in PSDP's action with PLDactivity.

The present results characterize PIPP remodeling as a rapid switch for“stop” signaling used by an extracellular regulator of PMN responses.LTB₄ receptor activation initiated a rapid and transient decrease inPSDP (FIG. 1) that coincided temporally with increased PLD activity(FIG. 2). As PSDP remodeling returned toward baseline values, PLDactivity decreased, revealing an inverse relationship and suggesting arole for PSDP in the regulation of this pivotal lipid-modifying enzyme.Cells exposed to LTB₄ and an LXA₄ receptor agonist (15-epi-LXa) showed adramatic switch in PSDP remodeling to give increased [³²P]-PSDP andmarked inhibition of both PLD activity and superoxide anion generation(FIGS. 1 & 2). In addition, synthetic PSDP was a selective and potentinhibitor of isolated PLD (FIG. 3, Table I), a property not shared byother closely related lipids. Taken together, the reciprocalrelationship between PSDP levels and PLD activity as well as directinhibition of recombinant human PLD1 b, purified cPLD and PLD activityin PMN lysates support a role for PSDP as an endogenous lipid regulatorof PMN PLD activity. The different temporal profiles of PIPP remodelinginitiated upon receptor activation by PMN ligands with opposing actions(i.e., stimulation and inhibition) suggest that PIPP remodeling and PSDPitself may serve as important components in intracellular signaling, inparticular as “stop” signals.

Cholesterol is not a biosynthetic product in PMN, as they lack a mixedfunction oxidase and cyclase necessary for its endogenous formation fromacetate (Shechter, I., Fogelman, A. M., and Popjak, G. (1980) Adeficiency of mixed function oxidase activities in the cholesterolbiosynthetic pathway of human granulocytes. J. Lipid Res. 21, 277-283).In view of the present findings, the resultant biosynthetic terminationat squalene in PMN suggests that products such as squalene's directprecursor, PSDP, carries functions distinct from cholesterolbiosynthesis. Hence, it is likely that the PIPP signaling pathwayuncovered in human PMN may extend to other cell types. In addition todietary influences known to impact mevalonate and polyisoprenylphosphate biosynthesis, PSDP formation is also actively regulated bysoluble immune stimuli and growth factors (FIGS. 1B, C).Granulocyte/macrophage-colony stimulating factor, for example, increasesPSDP remodeling in PMN whereas the chemotactic peptide, fMLP, triggers(within seconds) rapid decrements in PSDP and reciprocal increments inPSMP that return to baseline within 5-10 minutes (Levy, B. D., Petasis,N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989). This time course of PIPPremodeling is similar in magnitude and extent to LTB₄-initiateddecrements in PSDP (FIGS. 1B & C) and correlates well with the timecourse of activating neutrophil responses such as O₂ ⁻ generation, whichis inhibited by PSDP. The presence of PSDP in peripheral blood PMNdespite their inability to generate cholesterol from endogenous sources,its rapid remodeling in response to receptor-mediated inflammatorystimuli of diverse classes of receptor agonist, and its ability toinhibit PLD activity and NADPH oxidase at nanomolar levels aresupportive evidence for a role for PSDP as a novel negativeintracellular signal. Thus, this newly uncovered PIPP signaling mightfunction to decrease negative signal levels, in contrast to thewell-appreciated phosphotidylinositol signaling pathways (reviewed inPettit, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., andWakelam, M. J. O. (1997) Diacylglycerol and phosphatidate generated byphospholipases C and D, respectively, have distinct fatty acidcompositions and functions. J. Biol. Chem. 272, 17354-17359) that, whenactivated, rapidly generate positive intracellular stimuli (e.g.,inositol trisphosphate, diacylglycerol & Ca²⁺).

Aspirin, the lead non-steroidal anti-inflammatory drug, also effectscholesterol biosynthesis by mechanisms that remain to be completelyelucidated (Rabinowitz, J. L., Baker, D. G., Villanueva, T. G., Asanza,A. P., and Capuzzi, D. M. (1992) Liver lipid profiles of adults takingtherapeutic doses of aspirin. Lipids 27, 311-314). Beyond itswell-appreciated inhibition of cyclooxygenase (COX), aspirin can piratethis system to set in place an anti-inflammatory circuit generating15-epi-LX, carbon 15-R-epimers of the natural 15-S-containing-LX, duringcell-cell interactions by aspirin-acetylated COX-2 and 5-lipoxygenase(FIG. 1A and Chiang, N., Takano, T., Clish, C. B., Petasis, N. A., Tai,H.-H., and Serhan, C. N. (1998) Aspirin-triggered 15-epi-lipoxin A₄(ATL) generation by human leukocytes and murine peritonitis exudates:development of a specific 15-epi-LXA₄ ELISA. J. Pharmacol Exper. Ther.287, 779-790). These aspirin-triggered LX carry anti-inflammatory andanti-proliferative properties (Claria, J., and Serhan, C. N. (1995)Aspirin triggers previously undescribed bioactive eicosanoids by humanendothelial cell-leukocyte interactions. Proc. Natl. Acad. Sci. 92,9475-9479; Serhan, C. N. (1997) Lipoxins and Novel Aspirin-Triggered15-epi-Lipoxins: A Jungle of Cell-Cell Interactions or a TherapeuticOpportunity? Prostaglandins 53, 107-137) and may mediate a component ofaspirin's beneficial therapeutic actions. As observed in the presentexperiments, LXA₄ receptor activation by a 15-epi-LX mimetic reversedPSDP remodeling initiated by LTB₄ receptors, leading to increases inPSDP levels (FIG. 1). Since the 15-epi-LXa inhibited both PLD activityand superoxide anion generation (FIG. 2), these results implicate PIPPremodeling as a component of the cellular basis for aspirin's inhibitionof excessive inflammatory responses. In addition to regulating LTB₄'sstimulatory actions, this novel mechanism of inhibition of LTB₄ receptorsignaling may also play broader roles in host defense, as this receptorwas recently identified as a co-receptor for HIV-1 (Owman, C.,Garzino-Demo, A., Cocchi, F., Popovic, M., Sabirsh, A., and Gallo, R.(1998) The leukotriene B₄ receptor functions as a novel type ofcoreceptor mediating entry of primary HIV-1 isolates into CD4-positivecells. Proc. Natl. Acad. Sci. 95, 9530-9534).

Hydrolysis of PC to PA by PLD appears crucial in transmembrane signalingby a wide range of receptor classes during PMN activation (Olson, S. C.,and Lambeth, J. D. (1996) Biochemistry and cell biology of phospholipaseD in human neutrophils. Chem. Phys. Lipids 80, 3-19). Both G-proteinlinked receptors and receptor tyrosine kinases activate PLD. Inleukocytes, several factors including PKCα (in a kinase-independentmanner) and increased intracellular calcium can activate PLD1 (Exton, J.H. (1997) New developments in phospholipase D. J. Biol. Chem. 272,15579-15582). FMLP-stimulated PLD activity in PMN is increased bymembrane association of the ADP-ribosylation factor (ARF) and smallGTPase RhoA (Fensome, A., Whatmore, J., Morgan, C., Jones, D., andCockcroft, S. (1998) ADP-ribosylation factor and Rho proteins mediatefMLP-dependent activation of phospholipase D in human neutrophils. J.Biol. Chem. 273, 13157-13164). Of considerable interest here, PSDPdirectly inhibited recombinant hPLD1b in the absence of regulatoryproteins (see Table I). These results suggest that PSDP may inhibit PLDat its catalytic center and is likely to act at other PLD isoforms, suchas PLD1a and PLD2 isoforms where the catalytic centers are conserved.PSDP's ability to serve as an endogenous inhibitor of PLD likely resultsfrom PSDP's unique three-dimensional and physical chemical propertieswhich might now serve as a template for the preparation of more potentPLD inhibitors by design to fulfill the structure activity relationshipuncovered here.

Regulation of PMN activation in complex host responses is controlled inpart by soluble mediators and, in particular, by autacoids with opposingactions (Serhan, C. N., Haeggstrom, J. Z., and Leslie, C. C. (1996)Lipid mediator networks in cell signaling: update and impact ofcytokines. FASEB J. 10, 1147-1158), such as LT and LX, that here gavemarkedly different profiles for PIPP remodeling (FIG. 1). In most celltypes, PSDP is appreciated as a biosynthetic intermediate in cholesterolproduction by microsomal squalene synthase, which catalyzes head-to-headcondensation of two FDP (Jarstfer, M. B., Blagg, B. S. J., Rogers, D.H., and Poulter, C. D. (1996) Biosynthesis of squalene. Evidence for atertiary cyclopropylcarbinyl cationic intermediate in the rearrangementof presqualene diphosphate to squalene. J. Amer. Chem. Soc. 118,13089-13090). Ligand-operated rapid remodeling of PSDP in PMN is likelyto occur in membranes in proximity to LTB₄ and LXA₄ receptors andsuggests a non-microsomal pool of PSDP that may result from 1) novelbiosynthetic and/or metabolic pathways or 2) intracellular traffickingof PIPP with proteins from endoplasmic reticulum to membrane domains.Incorporation of [³²P] from ATP into PSDP but not FDP (see Results) isfurther evidence in support of a novel route for PSDP formation in PMN.The present results suggest that PIPP remodeling is linked to cellsurface receptor activation and is involved in the intracellulartransmission of extracellular ligands with opposing biological actions.In the present working model, a “negative lipid signal” (i.e., PSDP) isheld at a set point, like a ratchet, in “resting” cells. Incomingpositive signals (LTB₄, fMLP, etc.) initiate the degradation andinactivation of this inhibitory lipid (e.g., remodeling PSDP to theinactive monophosphate species, PSMP) (FIG. 1A and ref 22). Thus, PIPPremodeling enables mounting of intracellular positive signals thatthreshold for activation of select cellular processes. This type ofsignaling may explain the selectivity and tight coupling required byagonists such as LTB₄ that stimulate highly specialized functionalresponses of PMN such as chemotaxis, granule mobilization and superoxideanion generation. The extent to which this model of cell signaling,namely receptor-initiated degradation of negative lipid signals, occurswith other receptors and cell types remains for further studies.

In summary, ligand-operated rapid remodeling of PIPPs in human PMN anddirect inhibition of PLD activity at nanomolar levels support a role forPSDP as an intracellular signal and provide novel intracellular targetsby which PSDP can regulate cellular responses (Levy, B. D., Petasis, N.A., and Serhan, C. N. (1997) Polyisoprenyl phosphates in intracellularsignalling. Nature 389, 985-989). Given the wide occurrence of PIPP andcritical role of PLD in the plant and animal kingdoms, PIPP remodelingand direct inhibition of PLD first established here in human PMN mayhave wider implications in cell signaling in other cell types andspecies (Martin, A., Saqib, K. M., Hodgkin, M. N., Brown, F. D., Pettit,T. R., Armstrong, S., and Wakelam, M. J. O. (1997) Role and regulationof phospholipase D signalling. Biochem. Soc. Trans. 25, 1157-1160; Bach,T. J. (1995) Some new aspects of isoprenoid biosynthesis in plants—areview. Lipids 30, 191-202). The present results are the first to showdirect inhibition of a phospholipase involved in signal transduction byan endogenous intracellular lipid and set forth a new paradigm forlipid-protein interactions in the control of cellular responses, namelyreceptor-initiated degradation of a repressor lipid, that is alsosubject to regulation by aspirin ingestion via the actions ofaspirin-triggered 15-epimer LX. Together, these results suggest thatPIPP signaling pathways might also be of interest in pharmacologicinterventions and specifically that the conformation of PSDP can serveas a template for design of novel inhibitors.

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One having ordinary skill in the art will appreciate further featuresand advantages of the invention based on the above-describedembodiments. Accordingly, the invention is not to be limited by what hasbeen particularly shown and described, except as indicated by theappended claims. All publications and references cited herein, includingthose in the background section, are expressly incorporated herein byreference in their entirety.

1. A method for treating phospholipase D (PLD) initiatedpolymorphoneutrophil (PMN) inflammation in a subject, comprisingadministering to the subject an effective anti-inflammatory amount of alipoxin analog having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R_(T), —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein R_(T) is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —SO₃H, a hydrogen atom, halogen, methyl,—OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched, and hydroxyl; wherein Q₁ is (C═O), SO₂ or(CN), provided when Q₁ is CN, then X is absent; wherein one of R₂ and R₃is a hydrogen atom and the other is (a) H; (b) an alkyl of 1 to 8 carbonatoms, inclusive, which may be a straight chain or branched; (c) acycloalkyl of 3 to 6 carbon atoms, inclusive; (d) an alkenyl of 2 to 8carbon atoms, inclusive, which may be straight chain or branched; or (e)R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is alkylene of 0 to6 carbons atoms, inclusive, which may be straight chain or branched andwherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched, provided when R_(b) is 0, then R_(b) is ahydrogen atom; wherein R₄ is (a) H; (b) an alkyl of 1 to 6 carbon atoms,inclusive, which may be a straight chain or branched; wherein R₅ is

wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof, such that PLD initiated polymorphoneutrophil(PMN) inflammation is treated in a subject.
 2. The method of claim 1,wherein said method is performed in vitro.
 3. The method of claim 1,wherein said method is performed in vivo.
 4. A method for treatingphospholipase D (PLD) initiated superoxide generation or degranulationin a subject, comprising administering to the subject an effectiveanti-PLD amount of a lipoxin analog having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi)substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO_(2,) —CN, —C(═O)—R_(T), —SO₃H, a hydrogen atom,halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl;(vii) a detectable label molecule; or (viii) a straight or branchedchain alkenyl of 2 to 8 carbon atoms, inclusive; wherein R_(T) is (i) ahydrogen atom; (ii) an alkyl of 1 to 8 carbons atoms, inclusive, whichmay be straight chain or branched; (iii) a cycloalkyl of 3 to 10 carbonatoms; (iv) an aralkyl of 7 to 12 carbon atoms; (v) phenyl; (vi)substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —SO₃H, a hydrogen atom, halogen, methyl,—OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein one of R₂ and R₃ is a hydrogen atomand the other is (a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive,which may be a straight chain or branched; (c) a cycloalkyl of 3 to 6carbon atoms, inclusive; (d) an alkenyl of 2 to 8 carbon atoms,inclusive, which may be straight chain or branched; or (e) R_(a)Q₂R_(b)wherein Q₂ is —O— or —S—; wherein R_(a) is alkylene of 0 to 6 carbonsatoms, inclusive, which may be straight chain or branched and whereinR_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may be straightchain or branched, provided when R_(b) is 0, then R_(b) is a hydrogenatom; wherein R₄ is (a) H; (b) an alkyl of 1 to 6 carbon atoms,inclusive, which may be a straight chain or branched; wherein R₅ is

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO_(2,) —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof, such that PLD initiated superoxide generationor granulation is treated in a subject.
 5. The method of claim 4,wherein said method is performed in vitro.
 6. The method of claim 4,wherein said method is performed in vivo.